The field of the present invention relates to the treatment of infectious disease and cancer by inducing disease fighting immune responses. The growing problem of new strains of pathogenic bacteria resistant to antibiotics, the limited range of compounds effective against chronic viral and fungal infections and shortage of effective anti-cancer treatments demonstrates the need for compounds that can enhance the host defence against these medical problems. This invention relates to novel charged molecules which stimulate immune responses by binding to the Hepreceptor, a novel active site in human ezrin which I have discovered. The preferred charged molecules are novel peptides with sequences identical to the Hepreceptor in human ezrin.
Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins which play structural and regulatory roles in a wide range of cell types. There is considerable evidence to indicate that ezrin regulates the structure of the cortical cytoskeleton to control cell surface topography. Ezrin adopts two main conformations: 1) a soluble folded form which is found in the cytoplasm and. 2) an unfolded and elongated form which is found attached to the cytoplasmic surface of the cell membrane particularly in conduction with microvilli and other activation related structures. The N terminal domain of the protein is attached to the cytoplasmic surface of the membrane while the C terminal part binds to the actin cytoskeleton. Ezrin is a tyrosine kinase substrate in T cells and is also tyrosine phosphorylated as a result of Epidermal Growth Factor (EGF) stimulation of the EGF receptor. The N terminal domain of ezrin in its extended conformation binds to the cytoplasmic tail of CD44 in the presence of PIP2. Ezrin also may bind to the cytoplasmic tail of ICAM-2. Ezrin is very sensitive to regulatory proteases such as calpain and is rapidly turned over during cell activation.
Anthony Bretscher, David Reczek and Mark Berryman (1997).
“Ezrin: a protein requiring conformational activation to link microfilaments to the plasma membrane in the assembly of cell surface structures” Journal of Cell Science 110: 3011-3018.
Detailed analysis of the secondary structure of ezrin shows that there are three main structural domains: an N terminal domain from amino acids 1 to 300, a highly charged alpha domain from amino acids 300 to 470 and C terminal domain from amino acids 470 to 585. Structural modelling suggests that the alpha domain is folded into two anti-parallel helices in the soluble globular form of ezrin although the location of the hinge has not been identified. In the model of the extended phosphorylated form, ezrin is attached to the inner surface of the cell membrane by the N terminal domain, the alpha domains of two ezrin molecules are paired into anti-parallel dimers and located below the cell surface membrane. In this extended form, ezrin is tyrosine phosphorylated at tyrosine 353 (Yp 353).
Ossi Turunen, Markku Sainio, Juha Jaaskelainen. Olli Carpen, Antti Vaheri (1998) “Structure-Function relationships in the ezrin family and the effect of rumor-associated point mutations in neurofibromatosis 2 protein” Biochimica el Biophysica Acta 1387: 1-16.
I disclosed in U.S. Pat. No. 5,773,573 that the fourteen amino acid peptide HEP1, (amino acid sequence of TEKKRRETEREKE, SEQ ID 28, identical to amino acids 324 -337 of human ezrin) which has a 70% identity to the C terminus of gp120 could inhibit HIV replication in vivo in man. At the time I believed that the observed anti-HIV effect of peptide HEP1 was due to the orally administered HEP1 inducing
TABLE 1Amino acids, three letter code, one letter codeand side chain chargesCHARGES ON AMINO ACID SIDE CHAINS ATPHYSIOLOGICAL pHThreeOneletterletterAmino acidcodecodeChargeSymbolGlycineGlyGNONEAlanineAlaANONEValineValVNONEIsoleucineIleINONELeucineLeuLNONESerineSerSNONEThreonineThrTNONEAspartic acidAspDNEGATIVE−−Glutamic acidGluENEGATIVE−−PhosphotyrosineTyr(P)YpNEGATIVE−−AsparagineAsnNWEAK NEGATIVE−GlutamineGlnQWEAK NEGATIVE−LysineLysKPOSITIVE+ArginineArgRPOSITIVE+HistidineHisHWEAK POSITIVE+ProlineProPNONETryptophanTrpWNONEPhenylalaninePheFNONETyrosineTyrYNONEMethionineMetMNONECysteineCysCNONE